There has been considerable progress in the past decade on understanding the structure and function of T cell receptors (TCRs). The structures often a(3 TCR:peptide/MHC complexes have been solved and binding studies have shown that these interactions exhibit low affinities and slow on-rates. Structures and biophysical measurements indicate that the TCR has considerable flexibility, in the CDR3 loops in particular. Despite these studies, many important questions concerning T cell recognition and signaling still remain. For example, the energetic contribution of different peptide residues to TCR binding remains to be examined in a comprehensive way. Conversely, limited information is available on how regions of the TCR outside of the CDR3 loops contribute to peptide binding and specificity. It is also not at all clear what mechanism accounts for the conserved diagonal docking mode of TCRs. Related to this, the molecular basis of MHC restriction and alloreactivity for distinctly different MHC alleles are not understood (e.g. to what extent are specific TCR residues and/or TCR flexibility involved in these processes?). Finally, it remains enigmatic whether the a(3 TCR undergoes conformational changes in pepMHC distal regions upon pepMHC binding, and if so, if this movement contributes to signaling. We have developed a yeast display tec hnology to produce stabilized, higher affinity TCRs that will allow us to explore these questions. The project will use the class I restricted 2C TCR that recognizes, as strong agonists, a syngeneic complex SIYR/Kb and an allogeneic com plex QL9/Ld. We will also use the class II restricted TCR 3.L2 that recognizes Hb/l-Ek. The 3.L2 TCR was recently engineered, in collaboration with Paul Allen's lab, to produce high-affinity mutants, enabling crystallographic analysis of a 3.L2 TCR mutant:Hb/l-Ek complex (with Daved Fremont). The specific aims of the project are: 1) To measure [unreadable] the contribution of individualpeptide residues to binding by selected high-affinity TCR mutants. Using a high-affinity TCR against SIYR/Kb (KD = 16 nM), QL9/Ld (KD = 6 nM), and Hb/l-Ek (KD = 25 nM), we will determine the energetic contribution of each peptide residue. 2) To characterize the structure, flexibility, and binding mechanisms of selected TCR mutants. The wild type 2C:QL9/Ld alloant igen complex and several high-affinity TCRs (liganded and unliganded) will be examined structurally (with Chris Garcia);various binding studies will explore the mechanisms of the interactions. 3) To explore the role of TCRextracellular domain interactions in T cell signaling. Mutations at V:C and Va:Vp interfaces will be introduced into a T cell transfection system to examine their possible influence on T cell activation.